Metabolomic identification of α-ketoglutaric acid elevation in pediatric chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is the most common cause for non-relapse mortality postallogeneic hematopoietic stem cell transplant (HSCT). However, there are no well-defined biomarkers for cGVHD or late acute GVHD (aGVHD). This study is a longitudinal evaluation of metabolomic patterns of cGVHD and late aGVHD in pediatric HSCT recipients. A quantitative analysis of plasma metabolites was performed on 222 evaluable pediatric subjects from the ABLE/PBMTC1202 study. We performed a risk-assignment analysis at day + 100 (D100) on subjects who later developed either cGVHD or late aGVHD after day 114 to non-cGVHD controls. A second analysis at diagnosis used fixed and mixed multiple regression to compare cGVHD at onset to time-matched non-cGVHD controls. A metabolomic biomarker was considered biologically relevant only if it met all 3 selection criteria: (1) P ≤ .05; (2) effect ratio of ≥1.3 or ≤0.75; and (3) receiver operator characteristic AUC ≥0.60. We found a consistent elevation in plasma α-ketoglutaric acid before (D100) and at the onset of cGVHD, not impacted by cGVHD severity, pubertal status, or previous aGVHD. In addition, late aGVHD had a unique metabolomic pattern at D100 compared with cGVHD. Additional metabolomic correlation patterns were seen with the clinical presentation of pulmonary, de novo, and progressive cGVHD. α-ketoglutaric acid emerged as the single most significant metabolite associated with cGVHD, both in the D100 risk-assignment and later diagnostic onset analysis. These distinctive metabolic patterns may lead to improved subclassification of cGVHD. Future validation of these exploratory results is needed. This trial was registered at www.clinicaltrials.gov as #NCT02067832.

Effects of Co-Ingestion of ß-Hydroxy-ß-Methylbutyrate and L-Arginine α-Ketoglutarate on Jump Performance in Young Track and Field Athletes.

The aim of the study was to determine the effect of simultaneous supplementation of ß-hydroxy-ß-methylbutyrate and L-Arginine α-ketoglutarate on lower limb power and muscle damage in medium distance runners aged 15.3 (±0.9) years old. METHODS: The study group consisted of 40 volunteers aged 14-17 years practicing medium distance running for at least two years. The study lasted 12 days and followed a randomized, double-blind, placebo-controlled, parallel design. All subjects attended a familiarization session on day 0 before the test. The subjects were randomly divided into two groups: supplements and placebo group. The same training cycle protocol was used in both groups during the 12-day training period. Morning warm-up involved 10 min jogging at 60-75% of maximal heart rate and countermovement jump height measurement. Main training units were carried out for both groups with the same volume. Training load assessment (the daily session Rating of Perceived Exertion (s-RPE) method) method takes into consideration the intensity and the duration of the training session to calculate the "training load" (TL). RESULTS: At the end of the training cycle, a significant (p = 0.002) decrease in the countermovement jump (CMJ) height was found in the placebo group when compared to the baseline. In the supplement group, there was no decrease in the countermovement jump height. Creatine kinase and lactate dehydrogenase concentration increased during the training days similarly in both groups and decreased on rest days. There were no differences between groups in enzymes concentration. The research results indicate that the supplement combination used in the supplements group prevented a reduction in the CMJ values. In contrast to the supplements group, in the placebo group, the CMJ changes were statistically significant: a noticeable (p = 0.002) decrease in CMJ was noted between the baseline measurement and the 6th measurement. The well-being of the subjects from both groups changed significantly during the training period, and the intergroup differences in the mood level were similar and not statistically significant. CONCLUSIONS: The results of this study indicate that the daily co-supplementation with calcium salt of ß-hydroxy-ß-methylbutyrate (7.5 g) and L-Arginine α-ketoglutarate (10 g) during training might help to prevent decline in jump performance. No influence on muscle damage markers or mood was shown.

Identification of potential metabolic biomarkers of rectal cancer and of the effect of neoadjuvant radiochemotherapy.

We report a pilot study on the feasibility of determinations of circulating levels of paraoxonase-1 (PON1) and compounds related to energy metabolism as biomarkers for the evaluation of patients with rectal cancer (RC), and the effects produced by neoadjuvant radiochemotherapy (NRCT). We studied 32 patients treated with radiotherapy plus capecitabine concomitant chemotherapy and 48 control subjects. We identified pre-NRCT PON1 and α-ketoglutarate as the parameters that best discriminated between RC patients and the control group. Receiver operating characteristics analysis of the combination of the two parameters showed an area under the curve (AUC) of 0.918. Moreover, patients who presented a pathological complete response (pCR) to treatment had lower plasma pre-NRCT valine concentrations (AUC of 0.826). Patients who had a relapse had lower concentrations of succinate (AUC of 0.833). The results of the present study illustrate the usefulness of investigating alterations in oxidative stress and metabolism in RC. Due to the small number of patients studied, our results must be considered preliminary, but they suggest that the determination of circulating levels of PON1 and α-ketoglutarate might be a valuable tool for the early diagnosis of RC, while the determination of valine and succinate might effectively predict pCR and the appearance of relapse.

Dietary Alpha-Ketoglutarate Promotes Epithelial Metabolic Transition and Protects against DSS-Induced Colitis.

SCOPE: As a natural compound in foods, alpha-ketoglutarate (aKG) is one of the key metabolites maintaining energy homeostasis. This study examines the beneficial effects of dietary aKG against the development of experimental colitis and further explores the underlying molecular mechanisms. METHODS AND RESULTS: Eight-week-old male C57BL/6 mice receive drinking water with or without 1% aKG for 4 weeks. At week 3, colitis is induced by 2.5% dextran sulfate sodium (DSS) for 7 days followed by 7 days recovery. Dietary aKG supplementation decreases DSS-induced body weight loss, gross bleeding, fecal consistency score, and disease activity index. In agreement, aKG supplementation restores DSS-associated colon shortening, ameliorated mucosal damage, and macrophage infiltration into colonic tissue, which are associated with suppressed gut inflammation and Wnt signaling, and improved epithelial structure. Consistently, aKG supplementation enhances M1 to M2 macrophage polarization and strengthens intestinal barrier function. Additionally, aKG supplementation elevates colonic aKG levels while decreasing 2-hydroxyglutarate levels, which increases oxidative instead of glycolytic metabolism. CONCLUSION: aKG supplementation protects against epithelial damage and ameliorates DSS-induced colitis, which are associated with suppressed inflammation, Wnt signaling pathway, and glycolysis. Intake of foods enriched with aKG or aKG supplementation can be an alternative approach for the prevention or treatment of colitis that are common in Western societies.

Alpha-ketoglutarate ameliorates age-related osteoporosis via regulating histone methylations.

Age-related osteoporosis is characterized by the deterioration in bone volume and strength, partly due to the dysfunction of bone marrow mesenchymal stromal/stem cells (MSCs) during aging. Alpha-ketoglutarate (αKG) is an essential intermediate in the tricarboxylic acid (TCA) cycle. Studies have revealed that αKG extends the lifespan of worms and maintains the pluripotency of embryonic stem cells (ESCs). Here, we show that the administration of αKG increases the bone mass of aged mice, attenuates age-related bone loss, and accelerates bone regeneration of aged rodents. αKG ameliorates the senescence-associated (SA) phenotypes of bone marrow MSCs derived from aged mice, as well as promoting their proliferation, colony formation, migration, and osteogenic potential. Mechanistically, αKG decreases the accumulations of H3K9me3 and H3K27me3, and subsequently upregulates BMP signaling and Nanog expression. Collectively, our findings illuminate the role of αKG in rejuvenating MSCs and ameliorating age-related osteoporosis, with a promising therapeutic potential in age-related diseases.

Exercise-induced α-ketoglutaric acid stimulates muscle hypertrophy and fat loss through OXGR1-dependent adrenal activation.

Beneficial effects of resistance exercise on metabolic health and particularly muscle hypertrophy and fat loss are well established, but the underlying chemical and physiological mechanisms are not fully understood. Here, we identified a myometabolite-mediated metabolic pathway that is essential for the beneficial metabolic effects of resistance exercise in mice. We showed that substantial accumulation of the tricarboxylic acid cycle intermediate α-ketoglutaric acid (AKG) is a metabolic signature of resistance exercise performance. Interestingly, human plasma AKG level is also negatively correlated with BMI. Pharmacological elevation of circulating AKG induces muscle hypertrophy, brown adipose tissue (BAT) thermogenesis, and white adipose tissue (WAT) lipolysis in vivo. We further found that AKG stimulates the adrenal release of adrenaline through 2-oxoglutarate receptor 1 (OXGR1) expressed in adrenal glands. Finally, by using both loss-of-function and gain-of-function mouse models, we showed that OXGR1 is essential for AKG-mediated exercise-induced beneficial metabolic effects. These findings reveal an unappreciated mechanism for the salutary effects of resistance exercise, using AKG as a systemically derived molecule for adrenal stimulation of muscle hypertrophy and fat loss.

Potential osteomyelitis biomarkers identified by plasma metabolome analysis in mice.

Osteomyelitis, which often arises from a surgical-site infection, is a serious problem in orthopaedic surgery. However, there are no specific biomarkers for osteomyelitis. Here, to identify specific plasma biomarkers for osteomyelitis, we conducted metabolome analyses using a mouse osteomyelitis model and bioluminescence imaging. We divided adult male pathogen-free BALB/C mice into control, sham-control, and infected groups. In the infected group, a bioluminescent Staphylococcus aureus strain was inoculated into the femur, and osteomyelitis was detected by bioluminescence imaging. We next analysed the metabolome, by comprehensively measuring all of the small molecules. This analysis identified 279 metabolites, 12 of which were significantly higher and 45 were significantly lower in the infected group than in the sham-control and control groups. Principal component analysis identified sphingosine as the highest loading factor. Several acyl carnitines and fatty acids, particularly ω-3 and ω-6 polyunsaturated fatty acids, were significantly lower in the infected group. Several metabolites in the tricarboxylic acid cycle were lower in the infected group than in the other groups. Thus, we identified two sphingolipids, sphinganine and sphingosine, as positive biomarkers for mouse osteomyelitis, and two components in the tricarboxylic acid cycle, two-oxoglutarate and succinic acid, as negative biomarkers.

The metabolic fingerprints of HCV and HBV infections studied by Nuclear Magnetic Resonance Spectroscopy.

Few studies are available on metabolic changes in liver injuries and this is the first metabolomic study evaluating a group of HCV-positive patients, before and after viral eradication via DAA IFN-free regimens, using 1H-NMR to characterize and compare their serum fingerprints to naïve HBV-patients and healthy donors. The investigation clearly shows differences in the metabolomic profile of HCV patients before and after effective DAA treatment. Significant changes in metabolites levels in patients undergoing therapy suggest alterations in several metabolic pathways. It has been shown that 1H-NMR fingerprinting approach is an optimal technique in predicting the specific infection and the healthy status of studied subjects (Monte-Carlo cross validated accuracies: 86% in the HCV vs HBV model, 98.7% in the HCV vs HC model). Metabolite data collected support the hypothesis that the HCV virus induces glycolysis over oxidative phosphorylation in a similar manner to the Warburg effect in cancer, moreover our results have demonstrated a different action of the two viruses on cellular metabolism, corroborating the hypothesis that the metabolic perturbation on patients could be attributed to a direct role in viral infection. This metabolomic study has revealed some alteration in metabolites for the first time (2-oxoglutarate and 3-hydroxybutrate) concerning the HCV-infection model that could explain several extrahepatic manifestations associated with such an infection.

Association between admission plasma 2-oxoglutarate levels and short-term outcomes in patients with acute heart failure: a prospective cohort study.

BACKGROUND: 2-oxoglutarate (2OG), an intermediate metabolite in the tricarboxylic acid cycle, has been found to associate with chronic heart failure (HF), but its effect on short-term adverse outcomes in patients with acute HF (AHF) is uncertain. METHODS: This prospective cohort study included 411 consecutive hospitalized patients with AHF. During hospitalization, fasting plasma samples were collected within the first 24 h of admission. Plasma 2OG levels were measured by hydrophilic interaction liquid chromatography-liquid chromatography tandem mass spectrometry (HILIC-LC/MS/MS). All participants were followed up for six months. Multiple logistic regression was used to determine the odds ratio (OR) and 95% confidence interval (CI) for primary outcomes. RESULTS: The AHF cohort consisted of HF with preserved ejection fraction (EF) (64.7%), mid-range EF (16.1%), and reduced EF (19.2%), the mean age was 65 (±13) years, and 65.2% were male. Participants were divided into two groups based on median 2OG levels (µg/ml): low group (< 6.0, n = 205) and high group (≥6.0, n = 206). There was a relatively modest correlation between 2OG and N-terminal pro B-type natriuretic peptide (NT-proBNP) levels (r = 0.25; p < 0.001). After adjusting for age, sex, and body mass index, we found that the progression of the NYHA classification was associated with a gradual increase in plasma 2OG levels (p for trend< 0.001). After six months of follow-up, 76 (18.5%) events were identified. A high baseline 2OG level was positively associated with a short-term rehospitalization and all-cause mortality (OR: 2.2, 95% CI 1.3-3.7, p = 0.003), even after adjusting for NT-proBNP and estimated glomerular filtration rate (eGFR) (OR: 1.9, 95% CI 1.1-3.4, p = 0.032). After a similar multivariable adjustment, the OR was 1.4 (95% CI 1.1-1.7, p = 0.018) for a per-SD increase in 2OG level. CONCLUSIONS: High baseline 2OG levels are associated with adverse short-term outcomes in patients with AHF independent of NT-proBNP and eGFR. Hence plasma 2OG measurements may be helpful for risk stratification and treatment monitoring in AHF. TRIAL REGISTRATION: ChiCTR-ROC-17011240 . Registered 25 April 2017.

Succinate links atrial dysfunction and cardioembolic stroke.

OBJECTIVE: To determine whether altered metabolic profiles represent a link between atrial dysfunction and cardioembolic (CE) stroke, and thus whether underlying dysfunctional atrial substrate may contribute to thromboembolism risk in CE stroke. METHODS: A total of 144 metabolites were measured using liquid chromatography-tandem mass spectrometry in plasma samples collected within 9 hours of stroke onset in 367 acute stroke patients. Stroke subtype was assigned using the Causative Classification of Stroke System, and CE stroke (n = 181) was compared to non-CE stroke (n = 186). Markers of left atrial dysfunction included abnormal atrial function (P-wave terminal force in lead V1, PTFV1 >4,000 µV·ms), left atrial enlargement on echocardiography, and frank atrial fibrillation on ECG. Stroke recurrence risk was assessed using CHADS2 and CHA2DS2-VASc scores. Associations between metabolites and CE stroke, atrial dysfunction, and stroke recurrence risk were evaluated using logistic regression models. RESULTS: Three tricarboxylic acid metabolites-succinate (odds ratio [OR] 1.71, 95% confidence interval [CI] 1.36-2.15, p = 1.37 × 10-6), α-ketoglutarate (OR 1.62, 95% CI 1.29-2.04, p = 1.62 × 10-5), and malate (OR 1.58, 95% CI 1.26-1.97, p = 2.57 × 10-5)-were associated with CE stroke. Succinate (OR 1.36, 95% CI 1.31-1.98, p = 1.22 × 10-6), α-ketoglutarate (OR 2.14, 95% CI 1.60-2.87, p = 2.08 × 10-8), and malate (OR 2.02, 95% CI 1.53-2.66, p = 1.60 × 10-7) were among metabolites also associated with subclinical atrial dysfunction. Of these, succinate was also associated with left atrial enlargement (OR 1.54, 95% CI 1.23-1.94, p = 1.06 × 10-4) and stroke recurrence based on dichotomized CHADS2 (OR 2.63, 95% CI 1.68-4.13, p = 3.00 × 10-6) and CHA2DS2-VASc (OR 2.43, 95% CI 1.60-3.68, p = 4.25 × 10-6) scores. CONCLUSIONS: Metabolite profiling identified changes in succinate associated with CE stroke, atrial dysfunction, and stroke recurrence, revealing a putative underlying link between CE stroke and energy metabolism.

Novel circulating biomarkers for non-alcoholic fatty liver disease: A systematic review.

Currently, a liver biopsy remains the only reliable way to precisely diagnose non-alcoholic fatty liver disease (NAFLD) and establish the severity of liver injury, presence of fibrosis, and architecture remodeling. However, the cost and the intrinsic invasive procedure of a liver biopsy rules it out as a gold standard diagnostic test, and the imaging test are not the best choice due to the price, and currently is being refined. The lack of a biomarker of NAFLD pushes to develop this new line of research. The aim of the present systematic review is to clarify and update all the NAFLD biomarkers described in the literature until recently. We highlight α-ketoglutarate and CK18-F as currently the best potential biomarker of NAFLD. However, due to methodological differences, we propose the implementation of international, multicenter, multiethnic studies with larger population size, and biopsy proven NAFLD diagnosis to analyze and compare α-ketoglutarate and CK18-F as potential biomarkers of the silent evolution of NAFLD.

Effect of Diabetes on the Assessment Role of 2-Oxoglutarate to the Severity of Chronic Heart Failure.

BACKGROUND: Serum 2-oxoglutarate can reflect the severity of chronic heart failure (CHF) in patients without diabetes. Whether this predictive role persists in type 2 diabetes mellitus (T2DM) patients is unclear. In this study, we investigated this predictive role in T2DM patients and whether 2-oxoglutarate can indicate the diastolic or systolic function of left ventricle. METHODS: One hundred eighty CHF patients (76 with T2DM) and 66 healthy controls were studied. 2-Oxoglutarate was assayed by liquid chromatography-mass spectrometry/mass spectrometry. Echocardiographic parameters, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and other parameters were measured. RESULTS: 2-Oxoglutarate was increased in CHF patients with or without T2DM compared with controls (both P<0.01). Patients with a lower left ventricular ejection fraction or a higher NT-proBNP or left ventricular end-diastolic volume index had higher levels of 2-oxoglutarate (median, 18.77 µg/mL versus 11.25 µg/mL; median, 14.06 µg/ml versus 9.39 µg/ml; median, 18.06 µg/mL versus 11.60 µg/mL, all P<0.05) in nondiabetic patients but not in T2DM patients. In multiple logistic regression analysis, NT-proBNP (OR=3.445, 95% CI=1.098 to 10.816, P=0.034) and left ventricular end-diastolic diameter (OR=2.544, 95% CI=1.033 to 6.268, P=0.042) were independently associated with increased 2-oxoglutarate in nondiabetic patients. CONCLUSIONS: The levels of 2-oxoglutarate can reflect the clinical severity of CHF in nondiabetic patients but not in those with T2DM, and it can be used as a potential indicator of the systolic dysfunction of the left ventricle.

Plasma metabolomics reveals a diagnostic metabolic fingerprint for mitochondrial aconitase (ACO2) deficiency.

Mitochondrial respiratory chain dysfunction has been identified in a number of neurodegenerative disorders. Infantile cerebellar-retinal degeneration associated with mutations in the mitochondrial aconitase 2 gene (ACO2) has been recently described as a neurodegenerative disease of autosomal recessive inheritance. To date there is no biomarker for ACO2 deficiency and diagnosis relies on genetic analysis. Here we report global metabolic profiling in eight patients with ACO2 deficiency. Using an LC-MS-based metabolomics platform we have identified several metabolites with affected plasma concentrations including the tricarboxylic acid cycle metabolites cis-aconitate, isocitrate and alpha-ketoglutarate, as well as phosphoenolpyruvate and hydroxybutyrate. Taken together we report a diagnostic metabolic fingerprint for mitochondrial aconitase 2 deficiency.

Exploring effects of remote ischemic preconditioning in a pig model of hypothermic circulatory arrest.

OBJECTIVES: During aortic and cardiac surgery, risks for mortality and morbidity are inevitable. Surgical setups involving deep hypothermic circulatory arrest (DHCA) are effective to achieve organ protection against ischemic injury. The aim of this study was to identify humoural factors mediating additive protective effects of remote ischemic preconditioning (RIPC) in a porcine model of DHCA. DESIGN: Twenty-two pigs were randomized into the RIPC group (n = 11) and the control group (n = 11). The RIPC group underwent four 5-minute hind limb ischemia-reperfusion cycles prior to cardiopulmonary bypass and DHCA. All animals underwent identical surgical procedures including 60 min DHCA at 18 °C. Blood samples were collected from vena cava and sagittal sinus at several time points. After the 8-hour follow-up period, the brain, heart, and kidney tissue samples were collected for tissue analyses. RESULTS: Serum levels of brain damage marker S100B recovered faster in the RIPC group, after 4 hours of the arrest, (p < .05). Systemic lactate levels were lower and cardiac index was higher in the RIPC group postoperatively. Immunohistochemical cerebellum regional scores of antioxidant response regulator Nrf2 were better in the RIPC group (mean: 1.1, IQR: 0.0-2.5) compared with the control group (mean: 0.0, IQR: 0.0-0.0), reaching borderline statistical significance (p = .064). RIPC induced detectable modulations of plasma proteome and metabolites. CONCLUSIONS: The faster recovery of S100B, lower systemic lactate levels and favourable regional antioxidant response suggest possible neuronal cellular and mitochondrial protection by RIPC, whereas better cardiac index underlines functional effects of RIPC. The exact humoural factor remains unclear.

Circulating tricarboxylic acid cycle metabolite levels in citrin-deficient children with metabolic adaptation, with and without sodium pyruvate treatment.

Citrin deficiency causes adult-onset type II citrullinemia (CTLN-2), which later manifests as severe liver steatosis and life-threatening encephalopathy. Long-standing energy deficit of the liver and brain may predispose ones to CTLN-2. Here, we compared the energy-driving tricarboxylic acid (TCA) cycle and fatty acid ß-oxidation cycle between 22 citrin-deficient children (age, 3-13years) with normal liver functions and 37 healthy controls (age, 5-13years). TCA cycle analysis showed that basal plasma citrate and α-ketoglutarate levels were significantly higher in the affected than the control group (p<0.01). Conversely, basal plasma fumarate and malate levels were significantly lower than those for the control (p<0.001). The plasma level of 3-OH-butyrate derived from fatty acid ß-oxidation was significantly higher in the affected group (p<0.01). Ten patients underwent sodium pyruvate therapy. However, this therapy did not correct or attenuate such deviations in both cycles. Sodium pyruvate therapy significantly increased fasting insulin secretion (p<0.01); the fasting sugar level remained unchanged. Our results suggest that citrin-deficient children show considerable deviations of TCA cycle metabolite profiles that are resistant to sodium pyruvate treatment. Thus, long-standing and considerable TCA cycle dysfunction might be a pivotal metabolic background of CTLN-2 development.

Metabolite profiling can change health-care delivery to obese patients with fatty liver disease: the search for biomarkers.

Comorbidities associated with obesity have become a worldwide public health concern. Obesity-associated hepatic steatosis is not benign, and the risk of developing severe liver disease is high. Currently, biopsy is the only clinical tool available for the diagnosis of pathological alterations in the liver. However, the procedure is painful and not without risk. As such, there is a need to identify non-invasive biomarkers of steatosis. There has been considerable progress in this area, but research appears to be limited to measurements of levels of certain parameters in patients with liver impairment relative to those of healthy controls. The clinically relevant aim should be to distinguish, at an early stage, those obese individuals with liver steatosis from those obese individuals without it. Plasma constituents that act as surrogates of altered hepatic energy metabolism in response to food intake are likely candidates. Targeted metabolomics, combined with quantitation of the metabolites involved, has been shown to be an efficient measurement tool. Indeed, the evaluation of exhaled volatile compounds might be sufficient, while other rapid, sensitive, and reproducible methods have been validated in preliminary studies in various clinical settings. Metabolomics methods are promising but require considerable expertise and sophisticated (and expensive) equipment not readily available in all centers. The challenge is to adapt this newly acquired, expanding knowledge to current, reasonably equipped clinical laboratories, while substantially reducing costs. Good outcomes are urgently required if effective prevention programs are to be developed to decrease the prevalence of liver disease.

Increased Circulating Levels of Alpha-Ketoglutarate in Morbidly Obese Women with Non-Alcoholic Fatty Liver Disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) causes a wide spectrum of liver damage, ranging from simple steatosis to cirrhosis. However, simple steatosis (SS) and steatohepatitis (NASH) cannot yet be distinguished by clinical or laboratory features. The aim of this study was to assess the relationship between alpha-ketoglutarate and the degrees of NAFLD in morbidly obese patients. MATERIALS AND METHODS: We used a gas chromatography-quadruple time-of-flight-mass spectrometry analysis to quantify alpha-ketoglutarate in serum from normal-weight subjects (n = 30) and morbidly obese women (n = 97) with or without NAFLD. RESULTS: We found that serum levels of alpha-ketoglutarate were significantly higher in morbidly obese women than in normal-weight women. We showed that circulating levels of alpha-ketoglutarate were lower in lean controls and morbidly obese patients without NAFLD. We also found that alpha-ketoglutarate serum levels were higher in both SS and NASH than in normal liver of morbidly obese patients. However, there was no difference between SS and NASH. Moreover, we observed that circulating levels of alpha-ketoglutarate were associated with glucose metabolism parameters, lipid profile, hepatic enzymes and steatosis degree. In addition, diagnostic performance of alpha-ketoglutarate has been analyzed in NAFLD patients. The AUROC curves from patients with liver steatosis exhibited an acceptable clinical utility. Finally, we showed that the combination of biomarkers (AST, ALT and alpha-ketoglutarate) had the highest accuracy in diagnosing liver steatosis. CONCLUSION: These findings suggest that alpha-ketoglutarate can determine the presence of non-alcoholic fatty liver in morbidly obese patients but it is not valid a biomarker for NASH.

HPLC determination of α-ketoglutaramate [5-amino-2,5-dioxopentanoate] in biological samples.

α-Ketoglutaramate is an important glutamine metabolite in mammals, plants, and many bacteria. It is also a nicotine metabolite in certain bacteria. Previously published methods for the determination of α-ketoglutaramate in biological samples have considerable drawbacks. Here, we describe a relatively simple high-performance liquid chromatography (HPLC)-based method for measurement of α-ketoglutaramate in plasma and deproteinized tissues that overcomes these drawbacks. Concentrations of α-ketoglutaramate in normal rat liver, kidney, brain, and plasma were found to be approximately 216, 13, 6, and 19 µM, respectively. The HPLC method should be useful for studying the role of α-ketoglutaramate in eukaryotic glutamine metabolism and in bacterial nicotine metabolism.

Compound identification in gas chromatography/mass spectrometry-based metabolomics by blind source separation.

Metabolomics GC-MS samples involve high complexity data that must be effectively resolved to produce chemically meaningful results. Multivariate curve resolution-alternating least squares (MCR-ALS) is the most frequently reported technique for that purpose. More recently, independent component analysis (ICA) has been reported as an alternative to MCR. Those algorithms attempt to infer a model describing the observed data and, therefore, the least squares regression used in MCR assumes that the data is a linear combination of that model. However, due to the high complexity of real data, the construction of a model to describe optimally the observed data is a critical step and these algorithms should prevent the influence from outlier data. This study proves independent component regression (ICR) as an alternative for GC-MS compound identification. Both ICR and MCR though require least squares regression to correctly resolve the mixtures. In this paper, a novel orthogonal signal deconvolution (OSD) approach is introduced, which uses principal component analysis to determine the compound spectra. The study includes a compound identification comparison between the results by ICA-OSD, MCR-OSD, ICR and MCR-ALS using pure standards and human serum samples. Results shows that ICR may be used as an alternative to multivariate curve methods, as ICR efficiency is comparable to MCR-ALS. Also, the study demonstrates that the proposed OSD approach achieves greater spectral resolution accuracy than the traditional least squares approach when compounds elute under undue interference of biological matrices.